Identifying specific TLS-associated genes as potential biomarkers for predicting prognosis and evaluating the efficacy of immunotherapy in soft tissue sarcoma.

Background: The tertiary lymphatic structure (TLS) is an important component of the tumor immune microenvironment and has important significance in patient prognosis and response to immune therapy. However, the underlying mechanism of TLS in soft tissue sarcoma remains unclear. Methods: A total of 256 RNAseq and 7 single-cell sequencing samples were collected from TCGA-SARC and GSE212527 cohorts. Based on published TLS-related gene sets, four TLS scores were established by GSVA algorithm. The immune cell infiltration was calculated via TIMER2.0 and "MCPcounter" algorithms. In addition, the univariate, LASSO, and multivariate-Cox analyses were used to select TLS-related and prognosis-significant hub genes. Single-cell sequencing dataset, clinical immunohistochemical, and cell experiments were utilized to validate the hub genes. Results: In this study, four TLS-related scores were identified, and the total-gene TLS score more accurately reflected the infiltration level of TLS in STS. We further established two hub genes (DUSP9 and TNFSF14) prognosis markers and risk scores associated with soft tissue sarcoma prognosis and immune therapy response. Flow cytometry analysis showed that the amount of CD3, CD8, CD19, and CD11c positive immune cell infiltration in the tumor tissue dedifferentiated liposarcoma patients was significantly higher than that of liposarcoma patients. Cytological experiments showed that soft tissue sarcoma cell lines overexpressing TNFSF14 could inhibit the proliferation and migration of sarcoma cells. Conclusion: This study systematically explored the TLS and related genes from the perspectives of bioinformatics, clinical features and cytology experiments. The total-gene TLS score, risk score and TNFSF14 hub gene may be useful biomarkers for predicting the prognosis and immunotherapy efficacy of soft tissue sarcoma.

Efflux pumps as potential targets for biofilm inhibition.

Biofilms account for a great deal of infectious diseases and contribute significantly to antimicrobial resistance. Efflux pumps confer antimicrobial resistance to microorganisms and involve multiple processes of biofilm formation. Efflux pump inhibitors (EPIs) are attracting considerable attention as a biofilm inhibition strategy. The regulatory functions of efflux pumps in biofilm formation such as mediating adherence, quorum sensing (QS) systems, and the expression of biofilm-associated genes have been increasingly identified. The versatile properties confer efflux pumps both positive and negative effects on biofilm formation. Furthermore, the expression and function of efflux pumps in biofilm formation are species-specific. Therefore, this review aims to detail the double-edged sword role of efflux pumps in biofilm formation to provide potential inhibition targets and give an overview of the effects of EPIs on biofilm formation.

The effect of indoor air filtration on biomarkers of inflammation and oxidative stress: a review and meta-analysis of randomized controlled trials.

Improvement of indoor air quality is beneficial for human health. However, previous studies have not reached consistent conclusions regarding the effects of indoor air filtration on inflammation and oxidative stress. This study aims to determine the relationship between indoor air filtration and inflammation and oxidative stress biomarkers. We conducted an electronic search that evaluated the association of indoor air filtration with biomarkers of inflammation and oxidative stress in five databases (PubMed, Cochrane Library, EMBASE, Web of Science, and Scopus) from the beginning to April 23, 2023. Outcomes included the following markers: interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), C-reactive protein (CRP), malondialdehyde (MDA), 8-hydroxy-2deoxyguanosine (8-OHdG), and 8-iso-prostaglandinF2α (8-isoPGF2α). We extracted data from the included studies according to the system evaluation and the preferred reporting item for meta-analysis (PRISMA) guidelines and used the Cochrane risk of bias tool to assess bias risk. Our meta-analysis included 15 studies with 678 participants to assess the combined effect size. The meta-analysis demonstrated that indoor air filtration could have a marked reduction in IL-6 (SMD: -0.275, 95% CI: -0.545 to -0.005, p = 0.046) but had no significant effect on other markers of inflammation or oxidative stress. Subgroup analysis results demonstrated a significant reduction in 8-OHdG levels in the subgroup with < 1 day of duration (SMD: -0.916, 95% CI: -1.513 to -0.320; p = 0.003) and using filtrete air filter (SMD: -5.530, 95% CI: -5.962 to -5.099; p < 0.001). Our meta-analysis results depicted that indoor air filtration can significantly reduce levels of inflammation and oxidative stress markers. Considering the adverse effects of air pollution on human health, our study provides powerful evidence for applying indoor air filtration to heavy atmospheric pollution.

Branched chemically modified poly(A) tails enhance the translation capacity of mRNA.

Although messenger RNA (mRNA) has proved effective as a vaccine, its potential as a general therapeutic modality is limited by its instability and low translation capacity. To increase the duration and level of protein expression from mRNA, we designed and synthesized topologically and chemically modified mRNAs with multiple synthetic poly(A) tails. Here we demonstrate that the optimized multitailed mRNA yielded ~4.7-19.5-fold higher luminescence signals than the control mRNA from 24 to 72 h post transfection in cellulo and 14 days detectable signal versus <7 days signal from the control in vivo. We further achieve efficient multiplexed genome editing of the clinically relevant genes Pcsk9 and Angptl3 in mouse liver at a minimal mRNA dosage. Taken together, these results provide a generalizable approach to synthesize capped branched mRNA with markedly enhanced translation capacity.

N6-methyladenosine levels in peripheral blood RNA: a potential diagnostic biomarker for colorectal cancer.

BACKGROUND: N6-methyladenosine (m6A) is dysregulated in various cancers, including colorectal cancer (CRC). Herein, we assess the diagnostic potential of peripheral blood (PB) m6A levels in CRC. METHODS: We collected PB from healthy controls (HCs) and patients with CRC, analyzed PB RNA m6A levels and the expression of m6A-related demethylase genes FTO and ALKBH5, cocultured CRC cells with PB mononuclear cells (PBMCs), and constructed an MC38 cancer model. RESULTS: PB RNA m6A levels were higher in the CRC than that in HCs. The area under the curve (AUC) of m6A levels (0.886) in the CRC was significantly larger compared with carbohydrate antigen 199 (CA199; 0.666) and carcinoembryonic antigen (CEA; 0.834). The combination of CEA and CA199 with PB RNA m6A led to an increase in the AUC (0.935). Compared with HCs, the expression of FTO and ALKBH5 was decreased in the CRC. After coculturing with CRC cells, the PBMCs RNA m6A were significantly increased, whereas the expression of FTO and ALKBH5 decreased. Furthermore, m6A RNA levels in the PB of MC38 cancer models were upregulated, whereas the expression of FTO and ALKBH5 decreased. CONCLUSIONS: PB RNA m6A levels are a potential diagnostic biomarker for patients with CRC.

Design and rationale of the ATTRACTIVE trial: a randomised trial of intrAThrombus Thrombolysis versus aspiRAtion thrombeCTomy during prImary percutaneous coronary interVEntion in ST-segment elevation myocardial infarction patients with high thrombus burden.

INTRODUCTION: ST-segment elevation myocardial infarction (STEMI) with high thrombus burden is associated with a poor prognosis. Manual aspiration thrombectomy reduces coronary vessel distal embolisation, improves microvascular perfusion and reduces cardiovascular deaths, but it promotes more strokes and transient ischaemic attacks in the subgroup with high thrombus burden. Intrathrombus thrombolysis (ie, the local delivery of thrombolytics into the coronary thrombus) is a recently proposed treatment approach that theoretically reduces thrombus volume and the risk of microvascular dysfunction. However, the safety and efficacy of intrathrombus thrombolysis lack sufficient clinical evidence. METHODS AND ANALYSIS: The intrAThrombus Thrombolysis versus aspiRAtion thrombeCTomy during prImary percutaneous coronary interVEntion trial is a multicentre, prospective, open-label, randomised controlled trial with the blinded assessment of outcomes. A total of 2500 STEMI patients with high thrombus burden who undergo primary percutaneous coronary intervention will be randomised 1:1 to intrathrombus thrombolysis with a pierced balloon or upfront routine manual aspiration thrombectomy. The primary outcome will be the composite of cardiovascular death, recurrent myocardial infarction, cardiogenic shock, heart failure readmission, stent thrombosis and target-vessel revascularisation up to 180 days. ETHICS AND DISSEMINATION: The trial was approved by Ethics Committees of China-Japan Friendship Hospital (2022-KY-013) and all other participating study centres. The results of this trial will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT05554588.

Astaxanthin alleviates PM2.5-induced cardiomyocyte injury via inhibiting ferroptosis.

BACKGROUND: Long-term exposure of humans to air pollution is associated with an increasing risk of cardiovascular diseases (CVDs). Astaxanthin (AST), a naturally occurring red carotenoid pigment, was proved to have multiple health benefits. However, whether or not AST also exerts a protective effect on fine particulate matter (PM2.5)-induced cardiomyocyte damage and its underlying mechanisms remain unclear. METHODS: In vitro experiments, the H9C2 cells were subjected to pretreatment with varying concentrations of AST, and then cardiomyocyte injury model induced by PM2.5 was established. The cell viability and the ferroptosis-related proteins expression were measured in different groups. In vivo experiments, the rats were pretreated with different concentrations of AST for 21 days. Subsequently, a rat model of myocardial PM2.5 injury was established by intratracheal instillation every other day for 1 week. The effects of AST on myocardial tissue injury caused by PM2.5 indicating by histological, serum, and protein analyses were examined. RESULTS: AST significantly ameliorated PM2.5-induced myocardial tissue injury, inflammatory cell infiltration, the release of inflammatory factors, and cardiomyocyte H9C2 cell damage. Mechanistically, AST pretreatment increased the expression of SLC7A11, GPX4 and down-regulated the expression of TfR1, FTL and FTH1 in vitro and in vivo. CONCLUSIONS: Our study suggest that ferroptosis plays a significant role in the pathogenesis of cardiomyocyte injury induced by PM2.5. AST may serve as a potential therapeutic agent for mitigating cardiomyocyte injury caused by PM2.5 through the inhibition of ferroptosis.

Astaxanthin alleviates fine particulate matter (PM2.5)-induced lung injury in rats by suppressing ferroptosis and apoptosis.

Objectives: Fine particulate matter (PM2.5), a small molecule particulate pollutant, can reach the lungs via respiration and cause lung damage. Currently, effective strategies and measures are lacking to prevent and treat the pulmonary toxicity of PM2.5. Astaxanthin (ASX), a natural xanthophyll carotenoid, has attracted attention due to its unique biological activity. Our research aims to probe into the prevention and treatment of ASX on PM2.5-induced lung injury and clarify its potential mechanism. Methods: Sprague-Dawley (SD) rats were given olive oil and different concentrations of ASX orally daily for 21 days. PM2.5 suspension was instilled into the trachea of rats every two days for one week to successfully develop the PM2.5 exposure model in the PM2.5-exposed and ASX-treated groups of rats. The bronchoalveolar lavage fluid (BALF) was collected, and the content of lung injury-related markers was detected. Histomorphological changes and expression of markers associated with oxidative stress, inflammation, iron death, and apoptosis were detected in lung tissue. Results: PM2.5 exposure can cause changes in lung histochemistry and increase the expression levels of TP, AKP, ALB, and LDH in the BALF. Simultaneously, inflammatory responses and oxidative stress were promoted in rat lung tissue after exposure to particulate matter. Additionally, ASX preconditioning can alleviate histomorphological changes, oxidative stress, and inflammation caused by PM2.5 and reduce PM2.5-related ferroptosis and apoptosis. Conclusion: ASX preconditioning can alleviate lung injury after PM2.5 exposure by inhibiting ferroptosis and apoptosis.

Spatial atlas of the mouse central nervous system at molecular resolution.

Spatially charting molecular cell types at single-cell resolution across the 3D volume is critical for illustrating the molecular basis of brain anatomy and functions. Single-cell RNA sequencing has profiled molecular cell types in the mouse brain1,2, but cannot capture their spatial organization. Here we used an in situ sequencing method, STARmap PLUS3,4, to profile 1,022 genes in 3D at a voxel size of 194 × 194 × 345 nm3, mapping 1.09 million high-quality cells across the adult mouse brain and spinal cord. We developed computational pipelines to segment, cluster and annotate 230 molecular cell types by single-cell gene expression and 106 molecular tissue regions by spatial niche gene expression. Joint analysis of molecular cell types and molecular tissue regions enabled a systematic molecular spatial cell-type nomenclature and identification of tissue architectures that were undefined in established brain anatomy. To create a transcriptome-wide spatial atlas, we integrated STARmap PLUS measurements with a published single-cell RNA-sequencing atlas1, imputing single-cell expression profiles of 11,844 genes. Finally, we delineated viral tropisms of a brain-wide transgene delivery tool, AAV-PHP.eB5,6. Together, this annotated dataset provides a single-cell resource that integrates the molecular spatial atlas, brain anatomy and the accessibility to genetic manipulation of the mammalian central nervous system.

Construction of a Dendritic Nanoassembly-Based Fluorescent Biosensor for Electrostatic Interaction-Independent and Label-Free Measurement of Human Poly(ADP-ribose) Polymerase 1 in Lung Tissues.

Poly(ADP-ribose) polymerase 1 (PARP-1) is responsible for catalyzing the creation of poly(ADP-ribose) polymer and involved in DNA replication and repair. Sensitive measurement of PARP-1 is critical for clinical diagnosis. However, the conventional electrostatic attraction-based PAPR-1 assays usually involve laborious procedures, poor sensitivity, and false positives. Herein, we demonstrate the construction of a dendritic nanoassembly-based fluorescent biosensor for electrostatic interaction-independent and label-free measurement of human PARP-1 in lung tumor tissues. When PARP-1 is present, the specific double-stranded DNA (dsDNA)-activated PARP-1 transfers the ADP-ribosyl group from nicotinamide adenine dinucleotide (NAD+)/biotinylated NAD+ to the PARP-1 itself, resulting in the formation of biotinylated dsDNA-PARP-1-PAR polymer bioconjugates that can be captured by magnetic beads. Upon the addition of TdT, APE1, and NH2-modified T-rich probe, the captured dsDNAs with dual 3'-OH termini initiate TdT-activated APE1-mediated hyperbranched amplification to produce abundant dendritic DNA nanoassemblies that can be stained by SYBR Green I to generate a high fluorescence signal. This biosensor is characterized by a template-free, electrostatic interaction-independent, high sensitivity, and label-free assay. It enables rapid (less than 3 h) measurement of PARP-1 with a limit of detection of 4.37 × 10-8 U/µL and accurate measurement of cellular PARP-1 activity with single-cell sensitivity. Moreover, it is capable of screening potential inhibitors and discriminating the PARP-1 level in normal person tissues and lung cancer patient tissues, with great potential in PARP-1-related clinical diagnosis and drug discovery.

The evolution of mini-chromosomes in the fungal genus Colletotrichum.

Anthracnose diseases caused by Colletotrichum species are among the most common fungal diseases. These symptoms typically manifest as dark, sunken lesions on leaves, stems, and fruit. In China, mango anthracnose seriously affects fruit yield and quality. Genome sequencing of several species shows the presence of mini-chromosomes. These are thought to contribute to virulence, but their formation and activity remain to be fully elucidated. Here, we assembled 17 Colletotrichum genomes (16 isolated from mango plus one from persimmon) through PacBio long-read sequencing. Half of the assembled scaffolds had telomeric repeats at both ends indicating full-length chromosomes. Based on comparative genomics analysis at interspecies and intraspecies levels, we identified extensive chromosomal rearrangements events. We analyzed mini-chromosomes of Colletotrichum spp. and found large variation among close relatives. In C. fructicola, homology between core chromosomes and mini-chromosomes suggested that some mini-chromosomes were generated by recombination of core chromosomes. In C. musae GZ23-3, we found 26 horizontally transferred genes arranged in clusters on mini-chromosomes. In C. asianum FJ11-1, several potential pathogenesis-related genes on mini-chromosomes were upregulated, especially in strains with highly pathogenic phenotypes. Mutants of these upregulated genes showed obvious defects in virulence. Our findings provide insights into the evolution and potential relationships to virulence associated with mini-chromosomes. IMPORTANCE Colletotrichum is a cosmopolitan fungal genus that seriously affects fruit yield and quality of many plant species. Mini-chromosomes have been found to be related to virulence in Colletotrichum. Further examination of mini-chromosomes can help us elucidate some pathogenic mechanisms of Colletotrichum. In this study, we generated novel assemblies of several Colletotrichum strains. Comparative genomic analyses within and between Colletotrichum species were conducted. We then identified mini-chromosomes in our sequenced strains systematically. The characteristics and generation of mini-chromosomes were investigated. Transcriptome analysis and gene knockout revealed pathogenesis-related genes located on mini-chromosomes of C. asianum FJ11-1. This study represents the most comprehensive investigation of chromosome evolution and potential pathogenicity of mini-chromosomes in the Colletotrichum genus.

Spatially resolved single-cell translatomics at molecular resolution.

The precise control of messenger RNA (mRNA) translation is a crucial step in posttranscriptional gene regulation of cellular physiology. However, it remains a challenge to systematically study mRNA translation at the transcriptomic scale with spatial and single-cell resolution. Here, we report the development of ribosome-bound mRNA mapping (RIBOmap), a highly multiplexed three-dimensional in situ profiling method to detect cellular translatome. RIBOmap profiling of 981 genes in HeLa cells revealed cell cycle-dependent translational control and colocalized translation of functional gene modules. We mapped 5413 genes in mouse brain tissues, yielding spatially resolved single-cell translatomic profiles for 119,173 cells and revealing cell type-specific and brain region-specific translational regulation, including translation remodeling during oligodendrocyte maturation. Our method detected widespread patterns of localized translation in neuronal and glial cells in intact brain tissue networks.

Spatiotemporally resolved transcriptomics reveals the subcellular RNA kinetic landscape.

Spatiotemporal regulation of the cellular transcriptome is crucial for proper protein expression and cellular function. However, the intricate subcellular dynamics of RNA remain obscured due to the limitations of existing transcriptomics methods. Here, we report TEMPOmap-a method that uncovers subcellular RNA profiles across time and space at the single-cell level. TEMPOmap integrates pulse-chase metabolic labeling with highly multiplexed three-dimensional in situ sequencing to simultaneously profile the age and location of individual RNA molecules. Using TEMPOmap, we constructed the subcellular RNA kinetic landscape in various human cells from transcription and translocation to degradation. Clustering analysis of RNA kinetic parameters across single cells revealed 'kinetic gene clusters' whose expression patterns were shaped by multistep kinetic sculpting. Importantly, these kinetic gene clusters are functionally segregated, suggesting that subcellular RNA kinetics are differentially regulated in a cell-state- and cell-type-dependent manner. Spatiotemporally resolved transcriptomics provides a gateway to uncovering new spatiotemporal gene regulation principles.

Elafin is related to immune infiltration and could predict the poor prognosis in ovarian cancer.

Background: Ovarian cancer (OC) is the most lethal gynecologic malignancy, yet the clinical results for OC patients are still variable. Therefore, we examined how elafin expression affects the patients' prognoses and immunotherapy responses in OC, which may facilitate treatment selection and improve prognosis. Methods: The elafin mRNA expression profile was downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus. Elafin's prognostic potential and its relationship with clinical variables were investigated using Kaplan-Meier survival curves, time-dependent receiver operating characteristic curves as well as univariate and multivariate Cox regression models. As validation, protein expression in the tumor and adjacent tissues of OC patients was investigated by using immunohistochemistry (IHC). Comprehensive analyses were then conducted to explore the correlation between immune infiltration and elafin expression. Results: A higher mRNA expression of elafin was associated with an unfavorable prognosis in TCGA cohort and was validated in GSE31245 and IHC. Moreover, elafin was indicated as an independent risk factor for OC. A significantly higher protein expression of elafin was detected in the adjacent tissues of OC patients with shorter overall survival (OS). The immune-related pathways were mainly enriched in the high-elafin-mRNA-expression group. However, the mRNA expression of elafin was favorably correlated with indicators of the immune filtration and immunotherapy response, which also proved better immunotherapy outcomes. Conclusion: The high elafin expression was associated with an unfavorable OS, while it also indicated better immunotherapy responses. Thus, the detection of elafin is beneficial to diagnosis and treatment selection.

Integrative in situ mapping of single-cell transcriptional states and tissue histopathology in a mouse model of Alzheimer's disease.

Complex diseases are characterized by spatiotemporal cellular and molecular changes that may be difficult to comprehensively capture. However, understanding the spatiotemporal dynamics underlying pathology can shed light on disease mechanisms and progression. Here we introduce STARmap PLUS, a method that combines high-resolution spatial transcriptomics with protein detection in the same tissue section. As proof of principle, we analyze brain tissues of a mouse model of Alzheimer's disease at 8 and 13 months of age. Our approach provides a comprehensive cellular map of disease progression. It reveals a core-shell structure where disease-associated microglia (DAM) closely contact amyloid-ß plaques, whereas disease-associated astrocyte-like (DAA-like) cells and oligodendrocyte precursor cells (OPCs) are enriched in the outer shells surrounding the plaque-DAM complex. Hyperphosphorylated tau emerges mainly in excitatory neurons in the CA1 region and correlates with the local enrichment of oligodendrocyte subtypes. The STARmap PLUS method bridges single-cell gene expression profiles with tissue histopathology at subcellular resolution, providing a tool to pinpoint the molecular and cellular changes underlying pathology.

Mechanical stimulation on a microfluidic device to highly enhance small extracellular vesicle secretion of mesenchymal stem cells.

Small extracellular vesicles (sEVs) are recognized as promising detection biomarkers and attractive delivery vehicles, showing great potential in diagnosis and treatment of diseases. However, the applications of sEVs are usually restricted by their poor secretion amount from donor cells under routine cell culture conditions, which is especially true for mesenchymal stem cells (MSCs) due to their limited expansion and early senescence. Here, a microfluidic device is proposed for boosting sEV secretion from MSCs derived from human fetal bone marrow (BM-MSCs). As the cells rapidly pass through a microfluidic channel with a series of narrow squeezing ridges, mechanical stimulation permeabilizes the cell membrane, thus promoting them to secrete more sEVs into extracellular space. In this study, the microfluidic device demonstrates that mechanical-squeezing effect could increase the secretion amount of sEVs from the BM-MSCs by approximately 4-fold, while maintaining cellular growth state of the stem cells. Further, the secreted sEVs are efficiently taken up by immortalized human corneal epithelial cells and accelerate corneal epithelial wound healing in vitro, indicating that this technique wound not affect the functionality of sEVs and demonstrating the application potentials of this technique.

The prediction of in-hospital mortality in chronic kidney disease patients with coronary artery disease using machine learning models.

OBJECTIVE: Chronic kidney disease (CKD) patients with coronary artery disease (CAD) in the intensive care unit (ICU) have higher in-hospital mortality and poorer prognosis than patients with either single condition. The objective of this study is to develop a novel model that can predict the in-hospital mortality of that kind of patient in the ICU using machine learning methods. METHODS: Data of CKD patients with CAD were extracted from the Medical Information Mart for Intensive Care IV (MIMIC-IV) database. Boruta algorithm was conducted for the feature selection process. Eight machine learning algorithms, such as logistic regression (LR), random forest (RF), Decision Tree, K-nearest neighbors (KNN), Gradient Boosting Decision Tree Machine (GBDT), Support Vector Machine (SVM), Neural Network (NN), and Extreme Gradient Boosting (XGBoost), were conducted to construct the predictive model for in-hospital mortality and performance was evaluated by average precision (AP) and area under the receiver operating characteristic curve (AUC). Shapley Additive Explanations (SHAP) algorithm was applied to explain the model visually. Moreover, data from the Telehealth Intensive Care Unit Collaborative Research Database (eICU-CRD) were acquired as an external validation set. RESULTS: 3590 and 1657 CKD patients with CAD were acquired from MIMIC-IV and eICU-CRD databases, respectively. A total of 78 variables were selected for the machine learning model development process. Comparatively, GBDT had the highest predictive performance according to the results of AUC (0.946) and AP (0.778). The SHAP method reveals the top 20 factors based on the importance ranking. In addition, GBDT had good predictive value and a certain degree of clinical value in the external validation according to the AUC (0.865), AP (0.672), decision curve analysis, and calibration curve. CONCLUSION: Machine learning algorithms, especially GBDT, can be reliable tools for accurately predicting the in-hospital mortality risk for CKD patients with CAD in the ICU. This contributed to providing optimal resource allocation and reducing in-hospital mortality by tailoring precise management and implementation of early interventions.